Evidence of SARS-CoV-2 Transcriptional Activity in Cardiomyocytes of COVID-19 Patients without Clinical Signs of Cardiac Involvement

Abstract

Aims: A considerable proportion of patients affected by coronavirus respiratory disease (COVID-19) develop cardiac injury. The viral impact in cardiomyocytes deserves, however, further investigations, especially in asymptomatic patients.

Methods: We investigated for SARS-CoV-2 presence and activity in heart tissues of six consecutive COVID-19 patients deceased from respiratory failure showing no signs of cardiac involvement and with no history of heart disease. Cardiac autopsy samples were collected within 2 h after death, and then analysed by digital PCR, Western blot, immunohistochemistry, immunofluorescence, RNAScope, and transmission electron microscopy assays.

Results: The presence of SARS-CoV-2 into cardiomyocytes was invariably detected in all assays. A variable pattern of cardiomyocyte injury was observed, spanning from absence of cell death and subcellular alterations hallmarks, to intracellular oedema and sarcomere ruptures. In addition, we found active viral transcription in cardiomyocytes, by detecting both sense and antisense SARS-CoV-2 spike RNA.

Conclusions: In this autopsy analysis of patients with no clinical signs of cardiac involvement, the presence of SARS-CoV-2 in cardiomyocytes has been detected, determining variable patterns of intracellular damage. These findings suggest the need for cardiologic surveillance in surviving COVID-19 patients not displaying a cardiac phenotype.

Keywords: COVID-19; SARS-CoV-2; cardiomyocytes; heart.

Figure 1

RNA and protein of SARS-CoV-2 were present in cardiac samples of COVID-19 patients. (a) Chip-based digital PCR for SARS-CoV-2 N sequence in total RNA extracts from tissues of 3 out of 6 different patients and different cardiac regions. Raw expression values are expressed as log10 (copies/mL). The results are expressed as median and confidence interval (CI 95%). (b) Western blot analysis for ACE2, SARS-CoV-2 nucleoprotein, and spike protein in total protein extracts from tissues of 3 out of 6 different patients and different cardiac regions. GAPDH has been adopted as loading control. LV, left ventricle; Septum, interventricular septum; RV, right ventricle.

Figure 2

SARS-CoV-2 proteins were detectable in cardiomyocytes of COVID-19 patients. (a,b) Representative images of immunohistochemistry assays on 3 μm slides of formalin-fixed, paraffin-embedded left ventricle specimens from healthy controls (Healthy Ctrl, left panels) and COVID-19 patients (central and right panels) for SARS-CoV-2 nucleoprotein ((a), in red) and spike protein ((b), in brown). (c) Representative images of immunofluorescence assay on 3 μm slides of formalin-fixed, paraffin-embedded left ventricle specimens from healthy controls (Healthy Ctrl, left panels) and COVID-19 patients (central and right panels) for SARS-CoV-2 nucleoprotein (green) and sarcomeric α-actin (α-SARC, red). Nuclei have been stained by Hoechst (blue). Magnification as per scale bars.

Figure 3

SARS-CoV-2 sense and antisense RNA were localized in cardiomyocytes of COVID-19 patients. (a,b) Representative images of spatially resolved viral RNA detection by RNAScope assay on 3 μm slides of formalin-fixed, paraffin-embedded left ventricle specimens from healthy control (Healthy Ctrl, left panels) and COVID-19 patients (right panels) for SARS-CoV-2 sense (a) and antisense (b) probes for spike protein RNA sequences. Fast Red dots indicate viral RNA presence (white arrows). Nuclei have been counterstained with haematoxylin. Magnification as per scale bars.

Figure 4

Several cardiomyocytes containing SARS-CoV-2 nucleoprotein display intracellular alterations, but not cell death signs. (a,b) Representative images on consecutive slides of immunohistochemistry assay for SARS-CoV-2 nucleoprotein (a) and PTAH staining ((b), sarcomere structures in violet/blue), performed on left ventricle specimens from COVID-19 patients. Black arrows indicate strongly immunoreactive interstitial cells for SARS-CoV-2 nucleoprotein (in red), with a morphology consistent with macrophages. (c,d) Representative images of TUNEL assay on 3 μm slides of formalin-fixed, paraffin-embedded left ventricle specimens from COVID-19 patients. Damaged DNA is marked in green; nuclei have been counterstained with Hoechst (blue). Magnification as per scale bars.

Figure 5

SARS-CoV-2 virions were localized within both normal and altered cardiomyocytes of COVID-19 patients. (a,c,e) Electron micrograph showing cardiomyocytes containing viral particles, compatible with crown-shaped SARS-CoV-2. The viral particle diameter (a) is about 116 nm. (b,d,f) High magnifications of red squared areas of panels a, c, and e, respectively. In all panels, asterisks indicate normal mitochondrial and sarcomere structures, while cytoplasmic and sarcomere alterations are indicated with hashes.